Abstract Ischemic cardiomyopathy and heart failure are the leading causes of combined morbidity and mortality in humans. Clinical management of patients with inherited and acquired forms of cardiomyopathy is challenging and limited by the scarcity of new therapies in the pipeline. This proposal's immediate focus is on mechanistic insights into sarcomere regulation and heart performance with the long-term goal to ultimately advance novel approaches to redress heart disease. This effort underscores the significant health relevance of this proposal. The cardiac sarcomere is a complex allosteric regulatory system that drives heart contraction, but has been significantly under studied as a therapeutic target for cardiac disorders. For decades, the cardiac field has considered intracellular Ca2+ handling as the key pathway regulating heart performance in health and disease. By contrast, less emphasis has been placed on the role of the sarcomere in governing heart performance, representing a significant gap and missed opportunity. Until now, a major limitation in the field has been the inability to directly monitor sarcomere activation in live cardiac myocytes. Sarcomere performance has been a black box in terms of detecting, in real time, myofilament activation in live intact myocytes. We leverage here a methodological breakthrough enabling real-time detection of sarcomere activation in live cardiac myocytes. This innovation will lead our mechanistically-driven studies on the function of the sarcomere in health and disease, including elucidating the roles of sarcomeric activating ligands. Using this FRET-based sarcomere activation platform, we are uniquely poised to define the physiological significance of the regulatory role of the sarcomere in live cells. In illuminating fundamental properties of cardiac sarcomere regulation using live cell recordings, we are in a unique position to test the hypothesis that sarcomere function is uncoupled from Ca2+ handling in models of inherited and acquired cardiomyopathy, pointing to the sarcomere as a key target for experimental therapeutic discoveries. By the integration of three complementary Aims, we will determine the molecular mechanism underlying sarcomere-based excitation-contraction uncoupling and investigate the effects on live cell sarcomere activation due to disease causing mutations in myosin, troponin and small molecules in cardiac muscle. This proposal will advance a new understanding of heart performance regulation and open the door to potential breakthroughs in sarcomere modulating genes, proteins and small molecules to positively impact heart performance in health and disease.